This application relates generally to methods and apparatuses to obtain and analyze optically imaged samples including microbiological samples, and more specifically to methods and apparatuses for retaining at least one and preferably multiple randomly ordered microsphere arrays to solutions and to optical imaging systems for analysis.
It is known in the art to use probe arrays and sensors in systems to detect the presence and/or concentration of specific substances in fluids and gases. Many such systems rely on specific ligand/antiligand reactions as the detection mechanism. Pairs of substances (e.g., ligand and antiligands) are known to bind preferentially to each other, but to exhibit little or no binding with other substances.
Many prior art techniques utilize such binding pairs to detect complexes of interest. Often one component of the complex is labeled so as to make the entire complex detectable, using, for example, radioisotopes, fluorescent and other optically active molecules, enzymes, etc. Detection mechanisms utilizing luminescence are especially useful. Within the past decade, considerable development of optical fibers and fiber strands for use in combination with light absorbing dyes for chemical analytical determinations has occurred. The use of optical fibers for such purposes and techniques is described by Milanovich et al., xe2x80x9cNovel Optical Fiber Techniques For Medical Applicationxe2x80x9d, Proceedings of the SPIE 28th Annual International Technical Symposium On Optics and Electro-Optics, Volume 494, 1980; Seitz, W. R., xe2x80x9cChemical Sensors Based On Immobilized Indicators and Fiber Opticsxe2x80x9d in C.R.C. Critical Reviews In Analytical Chemistry, Vol. 19, 1988, pp. 135-173; Wolfbeis, O. S., xe2x80x9cFiber Optical Fluorosensors In 5 Analytical Chemistryxe2x80x9d in Molecular Luminescence Spectroscopy, Methods and Applications (S. G. Schulman, editor), Wiley and Sons, New York (1988); Angel, S. M., Spectroscopy 2 (4):38 (1987); Walt, et al., xe2x80x9cChemical Sensors and Microinstrumentationxe2x80x9d, ACS Symposium Series, Vol. 403, 1989, p. 252, and Wolfbeis, O. S., Fiber Optic Chemical Sensors, Ed. CRC Press, Boca Raton, Fla., 1991, 2nd Volume.
When using an optical fiber in an in vitro/in vivo sensor, at least one light absorbing dye is located near the fiber distal end. An appropriate source provides light, typically through the fiber proximal end, to illuminate the dye(s). As light propagates along the length of the optical fiber, a fraction of the propagated light exits the distal end and is absorbed by the dye. The light absorbing dye(s) may or may not be immobilized, may or may not be directly attached to the optical fiber itself, may or may not be suspended in a fluid sample containing one or more analyses of interest, and may or may not be retainable for subsequent use in a second optical determination.
Upon being dye absorbed, some light of varying wavelength and intensity returns to be conveyed through the same fiber or through collection fiber(s) to an optical detection system where it is observed and measured. Interactions between the light conveyed by the optical fiber and the properties of the light absorbing dye can provide an optical basis for both qualitative and quantitative determinations.
Many different classes of light absorbing dyes are conventionally employed with bundles of fiber strands and optical fibers for different analytical purposes. The more common dye compositions that emit light after absorption are termed xe2x80x9cfluorophoresxe2x80x9d, while dyes that absorb and internally convert light to heat (rather than emit as light) are termed xe2x80x9cchromophores.xe2x80x9d
Fluorescence is a physical phenomenon based upon the ability of some molecules to absorb light (photons) at specified wavelengths, and then emit light of a longer wavelength and at a lower energy. Substances able to fluoresce share a number of common characteristics: the ability to absorb light energy at one wavelength xcexab, reach an excited energy state, and subsequently emit light at another light wavelength xcexem. Absorption and fluorescence emission spectra are unique for each fluorophore and are often graphically represented as two slightly overlapping separate curves.
The same fluorescence emission spectrum is generally observed irrespective of the wavelength of the exciting light. Thus, within limits, the wavelength and energy of the exciting light may be varied, but the light emitted by the fluorophore will consistently exhibit the same emission spectrum. Finally, the strength of the fluorescence signal may be measured as the quantum yield of light emitted. The fluorescence quantum yield is the ratio of the number of photons emitted in comparison to the number of photons initially absorbed by the fluorophore. For more detailed information regarding each of these characteristics, the following references are recommended: Lakowicz, J. R., Principles of Fluorescence Spectroscopy, Plenum Press, New York, 1983; Freifelder, D., Physical Biochemistry, second edition, W. H. Freeman and Company, New York, 1982; xe2x80x9cMolecular Luminescence Spectroscopy Methods and Applications: Part Ixe2x80x9d (S. G. Schulman, editor) in Chemical Analysis, vol. 77, Wiley and Sons, Inc., 1985; The Theory of Luminescence, Stepanov and Gribkovskii, Iliffe Books, Ltd., London, 1968.
In contrast to fluorescence emitting materials, substances that absorb light but do not fluoresce usually convert the light into heat or kinetic energy. The ability to internally convert the absorbed light identifies the dye as a xe2x80x9cchromophore.xe2x80x9d Dyes that absorb light energy as chromophores do so at individual wavelengths of energy and are characterized by a distinctive molar absorption coefficient at that wavelength. Chemical analysis employing fiber optic strands, and absorption spectroscopy using visible and ultraviolet light wavelengths in combination with the absorption coefficient can determine concentration for specific analyses of interest using spectral measurement. The most common use of absorbance measurement via optical fibers is to determine concentration, which is calculated in accordance with Beers"" law. Accordingly, at a single absorbance wavelength, the greater the quantity of the composition that absorbs light energy at a given wavelength, the greater the optical density for the sample. In this fashion, the total quantity of light absorbed directly correlates with the quantity of the composition in the sample.
Many recent improvements in the use of optical fiber sensors in qualitative and quantitative analytical determinations concern the desirability of depositing and/or immobilizing various light absorbing dyes at the distal end of the optical fiber. In this manner, a variety of different optical fiber chemical sensors and methods have been reported for specific analytical determinations, and for applications such as pH measurement, oxygen detection, and carbon dioxide analyses. These developments are exemplified by the following publications: Freeman, et al., Anal Chem. 53:98 (1983); Lippitsch et al., Anal. Chem. Acta. 205: 1, (1988); Wolfbeis et al., Anal. Chem. 60:2028 (1988); Jordan, et al., Anal. Chem. 59:437 (1987); Lubbers et al.e, Sens. Actuators 1983; Munkholm et al., Talanta 35:109 10 (1988): Munkholmetal., Anal. Chem. 58:1427(1986); Seitz, W. R., Anal. Chem. 56:16A-34A (1984); Peterson, et al., Anal. Chem. 52:864 (1980): Saari, et al., Anal. Chem. 54:821 (1982); Saari, et al., Anal. Chem. 55:667 (1983); Zhujun et al., Anal. Chem. Acta. 160:47 (1984); Schwab, et al., Anal. Chem. 56:2199 (1984); Wolfbeis, O. S., xe2x80x9cFiber Optic Chemical Sensorsxe2x80x9d, Ed. CRC Press, Boca Raton, Fla., 1991, 2nd Volume; and Pantano, P., 15 Walt, D. R., Anal. Chem., 481A-487A, Vol. 67, (1995).
More recently, fiber optic sensors have been constructed that permit the use of multiple dyes with a single, discrete fiber optic bundle. For example, U.S. Pat. Nos. 5,244,636 , 5,250,264, and 5,320,814 to Walt et al. disclose systems for affixing multiple, different dyes on the distal end of a fiber optic bundle. Applicants refer to and incorporate herein by reference each said patent to Walt et al. The configurations disclosed in these patents to Walt et al. enable separate optical fibers of the bundle to optically access individual dyes. So doing avoids the problem of deconvolving the separate signals in the returning light from each dye. This problem can otherwise arise when signals from two or more dyes are combined, with each dye being sensitive to a different analyte, where there is significant overlap in the dyes"" emission spectra.
U.S. Pat. No. 6,023,540 and pending U.S. patent application Ser. No. 09/151,877 describe array compositions that utilize microspheres or beads on a surface of a substrate. Such substrate can be the terminal end of a fiber optic bundle, with each individual fiber comprising a bead containing an optical signature. Since the beads are deposited on the substrate surface randomly during fabrication, a unique optical signature is needed to xe2x80x9cdecodexe2x80x9d the array. Stated differently, after the array is fabricated, a correlation between location of an individual site on the array with the bead or bioactive agent at that particular site can be made. This implies that the beads may be randomly distributed on the array, an advantageously fast and inexpensive process when compared to an in situ synthesis or spotting techniques of the prior art. Once the array is loaded with the beads, the array may be decoded, or can be used with full or partial decoding occurring after testing.
A practical problem associated with the use of such probe arrays is how to properly retain and present the arrays to solutions and to optical imaging systems. Ideally each fiber optic bundle should be maintained parallel to each other, and normal to a holding mechanism to ensure accurate registration when optically imaging. Unfortunately prior art holding mechanisms do not readily meet this goal. Further, in prior art holders, if a fiber optic bundle becomes damaged or misaligned it is often necessary to discard the entire array of fiber optic bundle.
Thus there is a need for a mechanism to hold at least a single fiber optic bundle array and preferably a plurality of arrays in good registration. Such registration should be maintainable by the holder mechanism both during final machining of the fiber optic bundle ends, e.g., when the bundle ends are polished and loaded with beads or other analytic means, and during analysis. Preferably such mechanism should permit replacement of individual fiber optic bundles as may become necessary. Further, the holder mechanism should be straightforward and relatively inexpensive to produce
The present invention provides such holders and methods for using same.
In a first aspect, the present invention provides a holder defining at least one opening sized to engage and retain an end portion of a single fiber optic bundle or an array of such arrays. Each fiber optic bundle typically comprises a great many individual fiber optic strands that form an array. The holder typically is planar with spaced-apart upper and lower surfaces and may be made from metal, glass, ceramic, plastic, or epoxy (including thermosetting epoxy), among other materials. In cross-section, the fiber optic retaining opening is normal to a base plane of holder such that a length of fiber optic is retained in the holder perpendicular to the base plane. Preferably the holder will define an array of openings such that a plurality of fiber optic bundles may be retained in a preferred perpendicular orientation relative to the base. Since each retained bundle comprises many fiber optic strands, the holder is said to retain an array of arrays. The holder configuration ensures a proper registration relationship among the retained bundles, to solution containing wells and/or to an optical imaging system used to image the retained bundles. If desired, the holder may retain adjacent bundles such that multiple bundles can be processed within one well.
The opening in the base may be formed completely through the thickness of the holder, in which case a light source used for imaging may be presented from beneath the base or from above the base, at either the proximate or distal end of the retained fiber optic. On the other hand, if the optical system will be disposed above the base, the length of the base opening may be less than the base thickness. Individual bundles may be removed from the holder and replaced, if necessary, without discarding the entire array of arrays.
The holder may be sized similarly to a microscope slide, and the openings formed completely through the holder thickness. In this embodiment, the length of the retained fiber optics is made equal to this holder thickness such that the upper and lower end surface of each fiber optic bundle is respectively flush with the upper and lower surface of the holder. The holder, which may be formed from glass, or stainless steel, among other materials, may be imaged using a microscope, or scanning systems.
In an alternative embodiment, the bundle retaining openings are stepped to surround the retained bundle end with an annular region that is filled with a potting compound. Yet another embodiment surrounds each bundle retaining opening with at least one biased prong-like projection and preferably several such projections to bias or urge and to help maintain the retained bundle in an upright, perpendicular, disposition relative to a plane of the holder. In this embodiment the holder may, but need not, be formed from an injection molded plastic, and the retaining prong or prongs may be integrally molded with the base, or may be discrete components that are attached during formation of the holder.
In another embodiment, the array of fiber optic bundles are initially retained in proper registration in a temporary holder, and are then immersed in molten material such as wax, which is allowed to harden and form around the array of arrays. The hardened wax forms a semi-permanent fiber optic bundle holder, and the temporary holder is removed. The fiber optic bundles so retained by the wax may be lapped and polished, loaded with beads or otherwise treated, and placed into a target analyte-containing solution, and imaged.
The holder may be formed modularly as a laminated structure from planar modules that define grooves into which each bundle is inserted. A plurality of such modules are then assembled in sandwich fashion and held together with screws or the like. The distal ends of the retained bundles may be flush with or project from a face of the modular holder. Such modular holder may be used as an actual holder, or may be used as a temporary holder to facilitate placing bundle ends in a plate-like holder with bundle-retaining openings, or for securing the bundles in a wax holder as described herein.
Features from one holder embodiment may be combined with features from another embodiment. For example, prong-type holders may include through openings sized to frictionally retain a fiber optic bundle, which holes may include counter-bored regions or draft-angled surface regions, or conical shaped regions. These expanded regions can help align and retain the bundles before application of adhesive or potting compound to more permanently retain the bundles in the desired perpendicular registration. Alternatively, bundle ends may be press-fit, or retained within the holder using a controlled melting process.
An alternative embodiment provides a holder intended to retain a single bundle, which holder may be used in an optical imaging system. The holder is generally planar and includes a fixed member and a movable member that is biasedly urged to pivot towards the fixed member. When the two members are biased towards each other they define a gap sized to compressively retain a single fiber optic bundle from at least two sides. The form factor of the holder preferably is similar to that of embodiments intended to hold multiple bundles such that a single docking-type station may be used to retain each type of holder, for example during optical imaging.
Bundles retained in a holder according to the present invention are retained in registration suitable for exposing bundle ends to wells containing solutions, and/or for optical imaging, in situ or otherwise. Bundles may be attached to a holder, according to the present invention, at various process steps, including assembly of the array of arrays, before or after bundle end polishing, before or after bundle end etching, before or after insertion into test solutions, or before imaging or other analytical read-out process. In whatever mode of use, the holder provides a convenient and protective tool to retain an array of fiber optic bundles (or simply one such bundle) for handling, including handling during the various process and analytical steps. During the various process and analytic steps, the holder retains the bundles in a desired and consistent spatial registration, and further protects at least the retained bundle end from damage and from dust. The holder may be removed and reinserted into a docking station at various process steps, while consistently maintaining registration among the retained array of bundles. If desired, a surface of the holder may contain a barcode or other identification to uniquely identify the array of bundles that is retained.
Other features and advantages of the invention will appear from the following description in which the preferred embodiments have been set forth in detail, in conjunction with the accompanying drawings.